The folding of RNA molecules into specific tertiary structures is important for many cellular processes relating to gene expression: the protein synthesis machinery depends on intricate transfer and ribosomal RNA structures, processing of messenger and other RNAs requires specific structures, and the regulation of transcription and translation depend on the ability of RNA sequences to adopt specific structures in response to protein or metabolite signals. The chemical nature of RNA limits its structural possibilities; the negative charge causes sensitivity to the types and concentrations of ions present, and the high degree of hydration induces sensitivity to neutral solution components (osmolytes). The long term goals of the proposed studies are to investigate all the naturally-occurring solution factors that might influence RNA stability and identify the different strategies an RNA might use to accomplish a specific function in vivo. Specific questions will be investigated in each of three general areas: At a fundamental level, we will pursue our finding that some osmolytes stabilize RNA structures by affecting the hydration of RNA surfaces to obtain a more detailed picture of RNA hydration changes that accompany tertiary structure formation, and expand our studies of ions to include organic ions of in vivo relevance. Detailed studies will be made of the kinetics and energetics of conformational switches between alternative structures in two different RNAs in which the switch controls gene expression in response to changes in intracellular levels of free Mg2+ or a small metabolite (so-called riboswitch RNAs). These two RNAs use very different strategies to achieve stable tertiary structures, and probably function by different mechanisms as well; the studies are intended as explorations of these contrasting possibilities for functional RNAs. The physical studies of riboswitch RNAs will serve as a springboard for in vivo studies that will aim to elucidate the functional mechanism in the cellular environment, for example, how thermodynamic stability is balanced versus the kinetic rate of folding in determining functional response of RNA to cell signals, the reliance of RNAs on various ions found in vivo, and how changes in ion and osmolyte concentrations made by bacterial cells in response to changing growth conditions affect RNA function. These studies will help elucidate how RNAs function in a cellular environment, with potential applications in devising ways to block key RNA functions or design RNAs with specific regulatory functions. Bacterial pathogens are particularly reliant on riboswitch RNAs to survive in different environments inside and outside the human body, and are thus candidates for RNA-targeted therapeutics.